© The Author(s) 2020. Published by Oxford University Press For The Infectious Diseases Society of

Genomic diversity of SARS-CoV-2 in Coronavirus Disease 2019 patients
Zijie Shen 1,2#, Yan Xiao 3#, Lu Kang 1,2#, Wentai Ma 1,2#, Leisheng Shi 1,2, Li Zhang1,
Zhuo Zhou4, Jing Yang 1,2

, Jiaxin Zhong 1,2
, Donghong Yang5, Li Guo3, Guoliang
Zhang6, Hongru Li7, Yu Xu5, Mingwei Chen8, Zhancheng Gao5, Jianwei Wang 3, Lili
Downloaded from https://academic.oup.com/cid/article-abstract/doi/10.1093/cid/ciaa203/5780800 by guest on 16 April 2020

Ren 3*, Mingkun Li1,9*.
1. Key Laboratory of Genomic and Precision Medicine, Beijing Institute of

Genomics, Chinese Academy of Sciences, and China National Center for
Bioinformation, Beijing, 101300, China;
2. University of Chinese Academy of Sciences, Beijing 100049, China.
3. National Health Commission of the People’s Republic of China Key Laboratory
of Systems Biology of Pathogens and Christophe Mérieux Laboratory, Institute of
Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union
Medical College, Beijing 100730, China;
4. Biomedical Pioneering Innovation Center, Beijing Advanced Innovation Center
for Genomics, Peking-Tsinghua Center for Life Sciences, Peking University
Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene
Research, School of Life Sciences, Peking University, Beijing 100871, China
5. Department of Respiratory and Critical Care Medicine, Peking University
People's Hospital, Beijing 100044, China
6. National Clinical Research Center for Infectious Diseases, Guangdong Key
Laboratory for Emerging Infectious Diseases, Shenzhen Third People's Hospital,
Southern University of Science and Technology, Shenzhen 518112, China
7. Fujian Provincial Hospital, Fujian 350000, PR China
8. Department of Respiratory and Critical Care Medicine, the First Affiliated
Hospital of Xi'an Jiaotong University, Shaanxi Province 710061, P.R. China
© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of
America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
9. Center for Excellence in Animal Evolution and Genetics, Chinese Academy of
Sciences, Kunming, 650223, China;
# Author Z.S., Y.X., L.K., and W.M. contributed equally to this manuscript.
* Author L.R. and M.L. contributed equally to this manuscript.

Keywords: SARS-CoV-2, COVID-19, intra-host variant, microbiota, transmission
Summary
An elevated level of viral diversity was found in some SARS-CoV-2 infected patients,
indicating the risk of rapid evolution of the virus. Although no evidence for the
transmission of intra-host variants was found, the risk should not be overlooked.
Corresponding author:
Mingkun Li
E-mail: limk@big.ac.cn
Beijing Institute of Genomics, Chinese Academy of Sciences

No. 1-104, Beichen West Road, Chaoyang District, Beijing, 100101, China
Tel/Fax: 86-10-84097716
Alternate corresponding author:
Lili Ren
E-mail: renliliipb@163.com
Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union
Medical College
Building 7, Di Sheng Bei Jie Street 1, Yizhuang District, Beijing 100730, China;
Tel/Fax: 86-10-67837321
2
Abstract:
Background A novel coronavirus (SARS-CoV-2) has infected more than 75,000
individuals and spread to over 20 countries. It is still unclear how fast the virus evolved

and how the virus interacts with other microorganisms in the lung.
Methods We have conducted metatranscriptome sequencing for the bronchoalveolar
lavage fluid of eight SARS-CoV-2 patients, 25 community-acquired pneumonia (CAP)
patients, and 20 healthy controls.
Results The median number of intra-host variants was 1-4 in SARS-CoV-2 infected
patients, which ranged between 0 and 51 in different samples. The distribution of
variants on genes was similar to those observed in the population data (110 sequences).
However, very few intra-host variants were observed in the population as
polymorphism, implying either a bottleneck or purifying selection involved in the
transmission of the virus, or a consequence of the limited diversity represented in the
current polymorphism data. Although current evidence did not support the transmission
of intra-host variants in a person-to-person spread, the risk should not be overlooked.
The microbiota in SARS-CoV-2 infected patients was similar to those in CAP, either
dominated by the pathogens or with elevated levels of oral and upper respiratory
commensal bacteria.
Conclusion SARS-CoV-2 evolves in vivo after infection, which may affect its virulence,
infectivity, and transmissibility. Although how the intra-host variant spreads in the
population is still elusive, it is necessary to strengthen the surveillance of the viral
3
evolution in the population and associated clinical changes.
Abbreviations: COVID-19: Coronavirus disease 2019; CAP: Community-acquired
pneumonia; SARS: Severe acute respiratory syndrome; CoV: Coronavirus; Healthy:

Healthy controls; BALF: Bronchoalveolar lavage fluid; nCoV: COVID-19 patients; NC:
Negative controls.
4
Introduction
Since the outbreak of a novel coronavirus (SARS-CoV-2) in Wuhan, China, the
virus had spread to more than 20 countries, resulting in over 75,000 cases and more
than 2,300 deaths (Until Feb 22, 2020) [1, 2]. The basic reproduction number was

estimated to range from 2.2 to 3.5 at the early stage[3], making it a severe threat to
public health. Recent studies have identified bat as the possible origin of SARS-CoV-
2, and the virus likely uses the same cell surface receptor as SARS-CoV [4], namely
ACE2. These studies have advanced our understanding of SARS-CoV-2. However, our
knowledge of the novel virus is still limited.
The virus undergoes a strong immunologic pressure in humans, and may thus
accumulate mutations to outmaneuver the immune system [5]. These mutations could
result in changes in viral virulence, infectivity, and transmissibility [6]. Therefore, it is
imperative to investigate the pattern and frequency of mutations occurred. Aside from
the pathogen, microbiota in the lung is associated with disease susceptibility and
severity [7]. Alterations of lung microbiota could potentially modify immune response
against the viral and secondary bacterial infection [8, 9]. Thus, understanding the
microbiota, which comprises bacteria that could cause secondary infection or exert
effects on the mucosal immune system, might help to predict the outcome and reduce
complications.
In our study, we conducted metatranscriptome sequencing on bronchoalveolar
lavage fluid (BALF) samples from 8 subjects with Coronavirus disease 2019 (COVID-
19, the disease caused by SARS-CoV-2) patients. We found that the number of intra-
5
host variants ranged from 0 to 51 with a median number of 4, suggesting a high
evolution rate of the virus. By investigating a person-to-person spread event, we found
no evidence for the transmission of intra-host variants. Meanwhile, we found no
specific microbiota alteration in the BALF of COVID-19 patients comparing to CAP

patients with other suspected viral causes.
Results
Data summary
By metatranscriptome sequencing, more than 20 million reads were generated for
each BALF of COVID-19 patients (nCoV) as well as a negative control (nuclease-free
water, NC). For comparison, the metatranscriptome sequencing data with similar
number of reads from 25 virus-like community-acquired pneumonia patients (CAP,
determined by at least 100 viral reads and 10-fold higher than those in the NC), 20
healthy controls without any known pulmonary diseases (Healthy), and two extra NCs
(two saline solutions passing through the bronchoscope) were used in this study.
Demographic and clinical information was collected and summarized in Supplementary
Table 1.
After quality control, a median number of 55,571 microbial reads were generated
for each sample. nCoV had the highest proportion of microbial reads compared to CAP
and Healthy (nCoV: median proportion of 7%, CAP: 0.8%, Healthy:0.1%, p < 0.001,
Figure 1A), and 49% of the microbial reads could be mapped to SARS-CoV-2, which
was not different from the viral proportion in CAP (Figure 1B). Only SARS-CoV-2 was
identified in nCoV, and no read was mapped to other species belonging to
6
Betacoronavirus. Moreover, besides the detection of HCoV-OC43 in one Healthy and
HCoV-NL63 in a CAP, no other samples showed any signal of Betacoronavirus, which
proved the authenticity of the data and methods used in our analysis.
Intra-host variants in the genome of SARS-CoV-2

The sequencing depth of SARS-CoV-2 ranged from 18-fold in nCoV-5 to 32,291-
fold in nCoV1, with more than 80% of the genome covered by at least 50-fold in five
samples (Figure 2A, Supplementary Table 2). In total, 84 intra-host variants were
identified with minor allele frequency (MAF) greater than 5%, and 25 variants were
with MAF greater than 20% (Supplementary Table 3, Figure 2B, nCoV5 was excluded
from the analysis due to large gaps on its genome coverage). Notably, the number of
variants was not associated with the sequencing depth (Supplementary Figure 1). The
overall Ka/Ks ratio was significantly smaller than 1, which was similar for intra-host
variants and the polymorphisms observed in the population data, suggesting a purifying
selection acting on both types of mutations (Table 1). The numbers of variants observed
in the gene were proportional to gene lengths (cor = 0.950, p = 8E-06 for the intra-host
variant; cor = 0.957, p = 4E-06 for the polymorphisms). Although only a small fraction
of the variants was observed in multiple patients (2 out of 84, Figure 2C), some
positions were more prone to mutate or variants were transmitting in the population,
such as position 10779, where the mutant allele A was observed in all seven patients,
with the frequency ranging from 15% to 100% (Figure 2D).
The number of intra-host variants per individual showed a large variation (0 to 51,
median 4 for variants with MAF ≥ 5%; 0 to 19, median 1 for variants with MAF ≥ 20%),
7
which could not be explained by the batch effect, coverage variance, or contamination
(Supplementary Figure 1; nCoV1-4 were in one batch, nCoV5-8 were in another batch;
most mutations were not observed in the population data). We also noted that the
number of variations was not relevant to the days after symptom onset or the age of

patients (Supplementary Figure 2). Collectively, we did not find any reason for the
extremely high level of variants in nCoV6 (51 variants). A larger population size is
needed to investigate how frequent such outliers are, and whether they are associated
with the level of host immune response or the viral replication rate. We also noted
similar outliers for other viruses [11]. Of note, the origin of variants could be either
mutation occurred in vivo after infection or multiple transmitted SARS-CoV-2 strains.
No evidence for transmission of intra-host variants between samples
Among the eight COVID-19 patients, nCoV4 and nCoV7 were from the same
household, with dates of symptom onset differing by five days; thus a transmission from
nCoV4 to nCoV7 is highly suspected, especially considering that only nCoV4 had been
to the Huanan seafood market in Wuhan, which is the starting point of the outbreak and
suspected to be the source. First, the consensus sequence of the virus was the same for
two samples, and all four intra-host variants passing the selection criteria in nCoV4
were not detected in nCoV7 (Table 2). We further expanded the investigation to all
variants with MAF ≥ 2% and supported by at least 3 reads. By doing so, we detected
seven variants (out of 25) shared between the two samples. However, the MAF in both
nCoV4 and nCoV7 were similar to those in other samples, suggesting that these
positions were either error-prone or mutation-prone; hence they cannot support the
8
transmission of these variants.
Meanwhile, among all 84 intra-host variants, only three of them were found to be
polymorphic in the population data (position 7866 G/T; 27493 C/T; 28253 C/T). This
small number of overlap also suggests that intra-host variants were rarely transmitted

to other samples. However, we cannot rule out the possibility that the sequence diversity
in the population is underestimated by the current database.
Missing microbiota signature associated with SARS-CoV-2 infection
Metatranscriptome data also enabled us to profile the transcriptionally active
microbiota in different types of pneumonia, which is associated with the immunity
response in the lung [12, 13]. In general, a significant difference in microbiota
composition was observed among the nCoV, CAP, and Healthy groups (R2 = 0.07, p =
0.001; Figure 3A). However, the clustering of some samples with NC indicated a barren
microbiota in some samples. After removing the problematic samples and ambiguous
components, we still found that nCoV and CAP were both different from the healthy
controls (nCoV vs. Healthy: R2 = 0.45, p = 0.001; CAP vs. Healthy: R2 = 0.10, p =
0.002), implying a dysbiosis occurred in their lung microbiota. Microbiota could be
classified into three different types (Figure 3B). In particular, the microbiota in cluster
I was dominated by the possible pathogens, whereas the microorganisms in other
clusters were more diverse. By further inspecting the species belonging to each cluster
(Supplementary Table 4-5), we found that bacteria in Type III were mainly commensal
species frequently observed in the oral and respiratory tract, whereas bacteria in Type
II were mostly environmental organisms, thereby contamination was highly suspected.
9
Therefore, the microbiota was either pathogen-enriched (Type I) or commensal-
enriched (Type III) or undetermined due to low microbial load (Type II).
The microbiota in six nCoV samples were pathogen-enriched, and the other two
were commensal-enriched (Figure 3B). Moreover, two nCoV samples (2, 6) with an

excess number of intra-host SARS-CoV-2 variants both possessed the pathogen-
enriched microbiota. The overwhelming proportion of the virus may associate with a
higher replication rate, and could also potentially stimulate the intense immune
response against the virus, under which circumstance, an excess number of intra-host
mutations would be expected. However, as only eight nCoV patients were included in
this analysis, and the absolute microbial load was unknown, more data is needed for
further investigation.
Discussion
RNA viruses have a high mutation rate due to the lack of proofreading activity of
polymerases. Consequently, RNA viruses are prone to evolve resistance to drugs and
escape from immune surveillance. The mutation rate of SARS-CoV-2 is still unclear.
However, considering that the median number of pairwise sequence differences was 4
(Interquartile Range: 3-6) for 110 sequences collected between Dec 24, 2019 and Feb
9, 2020, the mutation rate should be at the same order of magnitude in SARS-CoV
(0.80-2.38×10-3 nucleotide substitution per site per year)[14]. The high mutation rate
also results in a high level of intra-host variants in RNA viruses [11, 15]. The median
number of intra-host variant in COVID-19 patients was 4 for variant with frequency ≥
5%, and this incidence was not significantly different from that reported in a study on
10
Ebola (655 variants with frequency ≥ 5% in 134 samples) (p>0.05)[11], suggesting that
the mutation rate of SARS-CoV-2 was also comparable to Ebola virus. An
exoribonuclease (ExoN) has been proposed to provide proofreading activity in SARS-
CoV[16, 17], and we noted that all three key motifs in the gene were identical between

SARS-CoV and SARS-CoV-2 (Supplementary Figure 3). In addition, neither
polymorphism nor intra-host variant was detected in these motifs, suggesting that the
gene is highly conserved, and thereby it could be a potential target for antiviral therapy.
Although we did not find any mutation hotspot genes in either polymorphism or intra-
host variants, the observation of shared intra-host variants among different individuals
implied the possibility of adaptive evolution of the virus in patients, which could
potentially affect the antigenicity, virulence, and infectivity of the virus [6].
It is worth noting that the SARS-CoV-2 genome in patients could be highly diverse,
which was also observed in other viruses [11]. The high diversity could potentially
increase the fitness of the viral population, making it hard to be eliminated[15]. Further
studies are needed to explore how this may influence the immune response towards the
virus and whether there is a selection acting on different strains in the human body or
during the transmission. In a single transmission event investigated in this study, we
found no evidence for the transmission of multiple strains. However, it is unclear
whether these intra-host variants occurred before the transmission or after the
transmission, which would result in different conclusions. Additionally, a bottleneck
may be involved in the transmission, which could also result in the loss of diversity
[18]. Nevertheless, the observation of high mutation burden in some patients
11
emphasized the possibility of rapid-evolving of this virus.
Recent studies have shown that the microbiota in the lung contributed to the
immunological homeostasis and potentially altered the susceptibility to viral infection.
Meanwhile, the lung microbiota could also be regulated by invading viruses [9, 19].

However, besides the feature that the microbial diversity was significantly lower in
pneumonia than that in healthy controls (Figure 3B), we did not identify any specific
microbiota pattern shared among COVID-19 patients, neither for CAP patients. A
possible reason for this could be the use of antibiotics in pneumonia patients. However,
this was not true for all pneumonia samples, as a substantial proportion of bacteria were
observed in some samples, including two COVID-19 patients. It is well known that a
common complication of viral infection, especially for respiratory viruses, secondary
bacterial infection often results in a significant increase in morbidity [20]. Thus, the
elevated level of bacteria in the BALF of some COVID-19 patients might increase the
risk of secondary infection. In the clinical data, the secondary infection rate for COVID-
19 was between 1%-10% [2, 21]. However, the quantitative relationship between
bacterial relative abundance/titer and infection is unclear.
Overall, our study has revealed the evolution of SARS-CoV-2 in the patient, a
common feature shared by most RNA viruses. How these variants influence the fitness
of viruses and genetic diversity in the population awaits further investigation. Currently,
only limited sequences are shared in public databases (Supplementary Table 6); hence
there is an urgent need to accumulate more sequences to trace the evolution of the viral
genome and associate the changes with clinical symptoms and outcomes.
12
Methods.
Subjects and samples collection
Eight COVID-19 pneumonia samples were collected from hospitals in Wuhan from

December 18 to 29, 2019; 25 virus-like community-acquired pneumonia (CAP)
samples were collected from Beijing Peking University People's Hospital, The
Shenzhen Third People's Hospital, Fujian Provincial Hospital, and The First-affiliated
hospital of Xi'an Jiaotong University between 2014 and 2018. CAP was diagnosed
following the guidelines of the Infectious Diseases Society of America and the
American Thoracic Society [22]. Pneumonia patients with chronic pulmonary diseases
were excluded. Meanwhile, BALF from 20 healthy volunteers were collected and used
as healthy controls. Demographic information and clinical information were included
in Supplementary Table 1.
For each patient, BALF samples were collected using a bronchoscope as part of
normal clinical management. The volume of BALF samples ranged between 5ml and
30ml, most of which were used for bacterial culture and the remnant were aliquoted
and stored at -80 ℃ before processing.
Metatranscriptome sequencing
A 200 ul aliquot of each SARS-CoV-2 infected whole-BALF sample was used to extract
RNA using Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA, USA) and Trizol
LS (Thermo Fisher Scientific, Carlsbad, CA, USA) in biosafety III laboratory, and the
rest samples were operated following the same protocol in biosafety II laboratory. The
13
RNA was then reverse transcribed, and amplified using an Ovation Trio RNA-Seq
library preparation kit (NuGEN, CA, USA) and was sequenced on an Illumina HiSeq
2500/4000 platform (Illumina, United Kingdom).
Data availability

The raw sequencing data reported in this paper have been deposited in the Genome
Warehouse in National Genomics Data Center [23], under project number
PRJCA002202 that is publicly accessible at https://bigd.big.ac.cn/gsa. Meanwhile, the
data have also been submitted to NCBI Sequence Read Archive (SRA) database under
project number PRJNA605907.
Data processing and taxonomic assignments
Quality control processes included adapter trimming, low quality reads removal, short
reads removal by fastp (-l 70, -x, --cut-tail, --cut_tail_mean_quality 20, version:
0.20.0)[24], low complexity reads removal by Komplexity (-F, -k 8, -t 0.2, version: Nov
2019)[25], host removal by bmtagger
(ftp://ftp.ncbi.nlm.nih.gov/pub/agarwala/bmtagger)[26, 27], and ribosomal reads
removal by SortMeRNA (version:2.1b)[28].
The resultant reads were mapped against NCBI nt database (version: Jul 1 2019)
using BLAST+ (version:2.9.0)(-task megablast, -evalue 1e-10, -max_target_seqs 10, -
max_hsps 1, -qcov_hsp_perc 60, -perc_identity 60)[29]. Taxonomic assignment was
done by MEGAN using lowest common ancestor algorithm (-ms 100, -supp 0, -me 0.01,
-top 10, -mrc 60, version: 6.11.0)[30]. After performing an overall PCoA and
Permanova test, samples and microorganisms were filtered for further analyses with the
14
following criteria. Samples with less than 5000 microbial reads were discarded.
Microorganisms satisfying the following criteria were considered in the microbiota
analysis, 1) archaea, bacteria, fungi, or virus; 2) with relative abundance ≥ 1% in the
raw data and filtered data; 3) supported by at least 100 reads; 4) abundance higher than

10-fold of that in the negative control; 5) no batch effect; 6) abundance was not
negatively correlated with bacteria titer; 7) not known contamination.
Intra-individual variants detection
Clean reads were mapped to the reference genome of SARS-CoV-2 (GenBank:
MN908947.3) using BWA mem (version:0.7.12)[31]. Duplicate reads were removed by
Picard (http://broadinstitute.github.io/picard; version: 2.18.22) [32] . Mpileup file was
generated by samtools (version 1.8)[33], and intra-host variants were called using
VarScan (version: 2.3.9)[34] and an in-house scripts. All variants had to satisfy the
following requirements: 1) Sequencing depth ≥ 50; 2) Minor allele frequency ≥ 5%; 3)
Minor allele frequency ≥ 2% on each strand; 4) Minor allele count ≥ 5 on each strand;
5) The minor allele was supported by the inner part of the read (excluding 10 bp on
each end); 6) Both alleles could be identified in at least 3 reads that specifically assigned
to genus Betacoronavirus.
For comparison with the polymorphism in the population, we obtained 110
sequences from GISAID (www.gisaid.org)[35, 36]. The accession number and
acknowledgment were included in Supplementary Table S6.
Statistical analysis.
Pearson’s chi-square test or Fisher’s exact test was used for categorical variables, and
15
the Mann-Whitney U test or Kruskal-Wallis rank sum test was used for continuous
variables that do not follow a normal distribution. A comparison of microbiota was done
by Permanova test.

Ethics statement.
The study was approved by the Institutional Review Board of Beijing Peking
University People's Hospital, The Shenzhen Third People's Hospital, Fujian
Provincial Hospital, and The First-affiliated hospital of Xi'an Jiaotong University. The
data collection for the COVID-19 patients were deemed by the National Health
Commission of the People’s Republic of China as the contents of the public health
outbreak investigation. Written informed consent was obtained from other pneumonia
patients and healthy controls.
Acknowledgments
We thank Dr. Xue Yongbiao and colleagues from National Genomics Data Center for
helpful discussion and computational resource support. We thank Dr. Huang Yanyi
(Peking University, Beijing, China) and Wang Jianbin (Tsinghua University, Beijing,
China) for providing the sequencing platform. We also thank Dr. Huang Chaolin for
assist in sample collection. We gratefully acknowledge the Authors, the Originating and
Submitting Laboratories for their sequence and metadata shared through GISAID, on
which some of our analysis is based, a full name list of all submitters was given in Table
S6.
16
Funding
This work was supported by grants from Innovation Fund for Medical Sciences [2016-
I2M-1-014], the National Major Science & Technology Project for Control and

Prevention of Major Infectious Diseases in China [2017ZX10103004,
2018ZX10305409, 2018ZX10301401, 2018ZX10732401]; National Natural Science
Foundation of China [31670169, 31871263]; and the Open Project of Key Laboratory
of Genomic and Precision Medicine, Chinese Academy of Sciences.
Potential conflicts of Interests
The authors declare no competing interests.
17
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37. Zhang Z, Li J, Zhao XQ, Wang J, Wong GK, Yu J. KaKs_Calculator: calculating Ka and
Ks through model selection and model averaging. Genomics Proteomics
Bioinformatics 2006; 4(4): 259-63.
38. Salter SJ, Cox MJ, Turek EM, et al. Reagent and laboratory contamination can critically
impact sequence-based microbiome analyses. BMC Biol 2014; 12: 87.
21
Table 1. The number of intra-host variants and polymorphisms in the genome of
SARS-CoV-2
P-
Intra-host variants Polymorphisms
value2
Gene length
NS S Ka/Ks1 NS S Ka/Ks
orf1a 13203 30 9 0.676 34 14 0.493* 0.627

orf1b 8088 8 5 0.355 10 8 0.277* 1
S 3822 8 3 0.599 10 6 0.375 0.692
ORF3a 828 2 1 0.561 4 2 0.561 1
E 228 0 0 NA 0 0 NA 1
M 669 2 0 NA 1 1 0.318 1
ORF6 186 1 0 NA 0 0 NA 1
ORF7a 366 2 0 NA 3 0 NA 1
ORF8 366 2 2 0.228 2 1 0.456 1
N 1260 7 2 1.048 5 7 0.214* 0.184
ORF10 117 0 0 NA 1 0 NA 1
Sum 29133 62 22 0.578* 70 39 0.368* 0.164
1.
Ka/Ks was calculated using KaKs_Calculator2.0 (MS model)[37], an asterisk is
added if Ka/Ks is significantly different from 1 (p < 0.05).
2.
P-value indicating whether a significant difference of Ka/Ks ratio was observed
between two types of mutations in the gene, Fisher Exact test.
22
Table 2. The allele frequency changes in transmission from nCoV4 to nCoV7
POS1 Ref_nCoV42 Alt_nCoV43 FRE Ref_nCoV7 Alt_nCoV7 FRE P-value4
376 119 177 0.598 9 0 0.000 0.0003
769 777 17 0.021 16 0 0.000 1
2037 1496 33 0.022 8 0 0.000 1
3290 2249 112 0.047 17 0 0.000 1

3306 1523 137 0.083 17 0 0.000 0.389
3321 1232 29 0.023 16 0 0.000 1
4511 685 26 0.037 11 0 0.000 1
4518 710 16 0.022 8 0 0.000 1
10771 1416 185 0.116 24 3 0.111 1
10773 1467 48 0.032 24 1 0.040 0.557
10779 987 401 0.289 18 8 0.308 0.829
10814 581 53 0.084 15 1 0.063 1
11387 653 15 0.022 4 0 0.000 1
13693 1237 38 0.030 9 0 0.000 1
15682 1321 46 0.034 8 1 0.111 0.269
15685 1342 47 0.034 8 1 0.111 0.270
18499 1783 108 0.057 19 0 0.000 0.621
18699 1013 46 0.043 12 0 0.000 1
21641 520 24 0.044 5 0 0.000 1
22270 2282 55 0.024 27 2 0.069 0.153
23127 1151 176 0.133 19 0 0.000 0.159
26177 492 11 0.022 6 0 0.000 1
27493 1535 1554 0.503 40 0 0.000 1.46E-12
28253 3600 487 0.119 34 0 0.000 0.028
29398 4671 127 0.026 47 0 0.000 0.636
Sum 34842 3968 0.102 421 17 0.028 1.45E-06
1.
The four intra-host variant positions with MAF≥5% and passing selection criteria
were highlighted in bold.
2.
Number of reads supporting the reference allele.
3.
Number of reads supporting the alternative (mutant) allele.
4.
P-value indicates whether the difference of allele frequency between nCoV4 and
nCoV7 is significant or not (Fisher Exact test).
23
Figure legends
Figure 1. Overview of the sequencing data. (A) The proportion of microbial reads in
different groups; (B) Proportion of the viral read in patients infected with different
viruses.
Figure 2. Intra-host variants in SARS-CoV-2 genome. (A) Genome coverage for

SARS-CoV-2. A dash line indicates coverage of 50; (B) Frequency distribution of all
intra-host variants, and the frequency of different mutations in polymorphism data was
shown on the right side; (C) Distribution of the intra-host variations and polymorphisms
on the genome of SARS-CoV-2. The outer ring displays the structure of the genome,
following by the polymorphisms distribution on the genome. The length of each bar
represents the number of sequences with this mutation. Due to a large variation of the
number (1-27), 5% of the bar length was added for each additional sequence. The inner
rings represent the distribution of intra-host variants in different patients (ID of each
patient was labeled on each ring). Red bar indicates a synonymous mutation, and blue
bar indicates a nonsynonymous mutation; (D) Frequency of the mutant allele at each
high level (with frequency ≥ 20%) intra-host variant position. Nucleotides at the
position (reference allele/alternative allele), mutation type (nonsynonymous,
synonymous, noncoding), gene name, amino acid change were labeled on the right side
of the heatmap. The total number of variants (with frequency ≥ 20%) in each sample
was labeled on top of the heatmap. The name of five samples with more than 80% of
the genome covered by at least 50-fold was labeled in blue. An open circle was added
if the sample had a sequencing depth less than 50-fold at this position.
Figure 3. Microbiota in the BALF of COVID-19 patients, CAP patients, and

healthy controls. A. Principal Coordinates Analysis (PCoA) of all samples. B.
Heatmap of microbiota composition after QC filter (filters were described in Methods).
The CAP samples were labeled as virus names followed by numbers. COVID-19
patients were highlighted by black rectangles, and two co-occurring bacterial clusters
were highlighted by red rectangles. The names of all viruses are labeled in blue, and
contaminant genera reported by Salter and colleagues are labeled in red[38].
24
25
Figure 1
26
Figure 2
27
Figure 3

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Genomic diversity of SARS-CoV-2 in Coronavirus Disease 2019 patients

Zhuo Zhou4, Jing Yang 1,2

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1. Key Laboratory of Genomic and Precision Medicine, Beijing Institute of

* Author L.R. and M.L. contributed equally to this manuscript.

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Beijing Institute of Genomics, Chinese Academy of Sciences

Alternate corresponding author:

Background A novel coronavirus (SARS-CoV-2) has infected more than 75,000

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Methods We have conducted metatranscriptome sequencing for the bronchoalveolar

lavage fluid of eight SARS-CoV-2 patients, 25 community-acquired pneumonia (CAP)

patients, and 20 healthy controls.

patients, which ranged between 0 and 51 in different samples. The distribution of

However, very few intra-host variants were observed in the population as

polymorphism, implying either a bottleneck or purifying selection involved in the

transmission of the virus, or a consequence of the limited diversity represented in the

of intra-host variants in a person-to-person spread, the risk should not be overlooked.

population is still elusive, it is necessary to strengthen the surveillance of the viral

Abbreviations: COVID-19: Coronavirus disease 2019; CAP: Community-acquired

pneumonia; SARS: Severe acute respiratory syndrome; CoV: Coronavirus; Healthy:

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Since the outbreak of a novel coronavirus (SARS-CoV-2) in Wuhan, China, the

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knowledge of the novel virus is still limited.

result in changes in viral virulence, infectivity, and transmissibility [6]. Therefore, it is

In our study, we conducted metatranscriptome sequencing on bronchoalveolar

evolution rate of the virus. By investigating a person-to-person spread event, we found

no evidence for the transmission of intra-host variants. Meanwhile, we found no

specific microbiota alteration in the BALF of COVID-19 patients comparing to CAP

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By metatranscriptome sequencing, more than 20 million reads were generated for

each BALF of COVID-19 patients (nCoV) as well as a negative control (nuclease-free

number of reads from 25 virus-like community-acquired pneumonia patients (CAP,

Demographic and clinical information was collected and summarized in Supplementary

identified in nCoV, and no read was mapped to other species belonging to

HCoV-NL63 in a CAP, no other samples showed any signal of Betacoronavirus, which

Intra-host variants in the genome of SARS-CoV-2

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with the frequency ranging from 15% to 100% (Figure 2D).

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mutation occurred in vivo after infection or multiple transmitted SARS-CoV-2 strains.

No evidence for transmission of intra-host variants between samples

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in the population is underestimated by the current database.

Missing microbiota signature associated with SARS-CoV-2 infection

Metatranscriptome data also enabled us to profile the transcriptionally active

microbiota in different types of pneumonia, which is associated with the immunity

response in the lung [12, 13]. In general, a significant difference in microbiota

0.002), implying a dysbiosis occurred in their lung microbiota. Microbiota could be

I was dominated by the possible pathogens, whereas the microorganisms in other

II were mostly environmental organisms, thereby contamination was highly suspected.

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the mutation rate of SARS-CoV-2 was also comparable to Ebola virus. An

exoribonuclease (ExoN) has been proposed to provide proofreading activity in SARS-

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during the transmission. In a single transmission event investigated in this study, we

found no evidence for the transmission of multiple strains. However, it is unclear

transmission, which would result in different conclusions. Additionally, a bottleneck

[18]. Nevertheless, the observation of high mutation burden in some patients

immunological homeostasis and potentially altered the susceptibility to viral infection.

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common complication of viral infection, especially for respiratory viruses, secondary